The present invention relates generally to the fields of genetics, cellular and molecular biology, and enzymology. More particularly, the invention relates to novel galactose oxidase enzymes, polynucleotides, and polypeptides.
The enzyme galactose oxidase (GO), encoded by the galactose oxidase gene (go) of Dactylium dendroides and other organisms, catalyzes a reaction in which a primary alcohol such as the C6 hydroxyl group of galactose is oxidized to an aldehyde with concomitant reduction of molecular oxygen to hydrogen peroxide, as shown generally in Formula I.
RCH2OH+O2xe2x86x92RCHO+H2O2xe2x80x83xe2x80x83(I) 
GO enzymes may be readily used to oxidize the D-galactose (and other hydroxyl-containing) side-chains of many complex compounds, including, but not limited to molecules comprising a D-galactose moiety that is not sterically hindered or blocked at the C6 hydroxyl, and molecules comprising a moiety such as dihydroxyacetone, glycerol, or similar short-chain alcohols in which a primary hydroxyl functional group is present. Of particular interest is the oxidation of guar gum. When guar gum is oxidized by GO, the resulting compound, called oxidized guar, can be used for many purposes, including use in paper manufacturing to add strength to paper products via the formation of acetal, hemiacetal, and other crosslinks with cellulose fibers (see, e.g., 1996; Aldehyde cationic derivatives of galactose containing polysaccharides used as paper strength additives; U.S. Pat. No. 5,554,745 (Chiu et al.); U.S. Pat. No. 5,502,091 (Dasgupta).
Galactose oxidases have been isolated from several species. For example, U.S. Pat. No. 6,090,604 discloses a genomic DNA sequence and deduced amino acid sequence for the GO enzyme from Fusarium venenatum. 
Wildtype GO enzymes, however, are relatively inefficient oxidizers of guar gum and other compounds. Thus, there exists a need in the art for superior oxidizers of such compounds. Additionally, one obstacle to the development of variant GO""s is the high viscosity of guar (e.g., the cationic guar used in this work has a viscosity of 1000 cps in 1% aqueous solution), a high molecular weight polymeric substrate. Indeed, many natural or synthetic polymers are insoluble or highly viscous when in solution, and are consequently difficult to pipette by hand or robotic means. Therefore, various methods of high-throughput screening used to evaluate such variant enzymes useful in adding functionality to viscous or insoluble polymers are needed by the art. Such methods enable those of skill in the art to create mutant GO enzymes capable of more efficient oxidative enzymatic reactions.
Although GO does display significant activity towards guar, the present inventors improved its specific activity via in vitro evolution of the enzyme. Using selected methods of mutagenesis, the present inventors created mutant galactose oxidase genes (mgo""s) which encode variant galactose oxidase enzymes (vGO""s), which variants are superior to wildtype GO in terms of efficiency of oxidizing guar and other compounds, as well as in conferring improved thermostability.
Error prone PCR (EPP) was used to generate mutant go genes, encoding variant GO enzymatic proteins. One of skill in the art will appreciate that any method capable of generating mutant genes would be suitable for practicing the present invention. In order to evaluate the efficiency of oxidation of the variants, certain recently developed methods of high throughput screening were used. However, one of skill in the art may choose a method of screening suitable to the substrate of interest. One aspect of the particular screening method used for the evolution of vGO""s by the present inventors is the use of a proxy, i.e., a substrate that represents an adequate substitute for a problematic compound. Particularly, in order to evaluate vGO""s oxidation of guar, the proxy methyl-xcex1-D-galactose (methyl galactose) was used. The variant GO""s of the present invention are demonstrably superior to wildtype GO in terms of their ability to oxidize guar and other complex compounds having hydroxyl-containing sidechains.
In one aspect, the present invention provides polynucleotides comprising mutant go genes. In another aspect, the invention provides polypeptides encoded by such polynucleotides. In another aspect, the invention provides variant GO enzymes having superior enzymatic activity (on methyl galactose or other substrates), and/or thermostability (i.e., resistance to heat inactivation), and which differ from wildtype GO by having at least one substituted amino acid. In another aspect, the present invention provides vectors comprising the polynucleotides. In another aspect, the present invention provides cells transfected or transformed with such vectors. In still another aspect, the present invention provides antibodies specific to the variant GOs. In another aspect, the present invention provides methods of using these molecules and constructs.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) encoding variant GO enzymes which differ from wildtype GO by at least one amino acid, and which exhibit superior enzymatic activity (on methyl galactose or other substrates) and/or thermostability. DNA polynucleotides of the invention include genomic DNA, cDNA, and DNA that has been chemically synthesized in whole or in part. The present invention also provides vectors comprising such polynucleotides, and cells transfected with such vectors. The present invention also provides the proteins encoded by such polynucleotides (i.e., variant GO enzymes), and methods of using the polynucleotides and polypeptides. The present invention also provides antibodies specific to vGOs capable of binding specifically to the variants while remaining unbound to wildtype GO.
Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.
xe2x80x9cSynthesizedxe2x80x9d as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. xe2x80x9cWhollyxe2x80x9d synthesized DNA sequences are therefore produced entirely by chemical means, and xe2x80x9cpartiallyxe2x80x9d synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. By the term xe2x80x9cregionxe2x80x9d is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. The term xe2x80x9cdomainxe2x80x9d is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.
As used herein, the term xe2x80x9cactivityxe2x80x9d refers to a variety of measurable indicia suggesting or revealing the extent of a catalytic reaction peformed by an enzyme, binding, either direct or indirect; affecting a response, i.e. having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event. The term xe2x80x9cbetterxe2x80x9d activity, or xe2x80x9cincreasedxe2x80x9d activity, or xe2x80x9csuperiorxe2x80x9d activity, or the like, means an activity compared to the wildtype (and measured under the same conditions) that is at least about 10% higher, preferably at least about 25% higher, more preferably at least about 50% higher, more preferably at least about 75% higher, more preferably at least about 100% higher, more preferably at least about 1.35-fold higher, more preferably at least about 2-fold higher, more preferably at least about 3-fold higher, and most preferably at least about 4-fold higher. Such better, increased, or superior activity may be exhibited at particular desirable conditions of temperature, pressure, solution contents, and the like. Enzymatic activity of a GO enzyme may be measured using Vmax/Km, as described below in Example 1. A variant GO enzyme of the present invention has a Vmax/Km greater than 0.005 xcex94OD405/min mM.
As used herein, the abbreviation in italicized lower case (go) refers to a gene, cDNA, RNA or nucleic acid sequence while the upper case version (GO) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence.
As used herein, the term xe2x80x9cantibodyxe2x80x9d is meant to refer to complete, intact antibodies, and Fab, Fabxe2x80x2, F(ab)2, and other fragments thereof. Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, polyclonal antibodies, chimeric antibodies, and humanized antibodies.
As used herein, the term xe2x80x9cbindingxe2x80x9d means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.
As used herein, the term xe2x80x9ccompoundxe2x80x9d means any identifiable chemical or molecule, including, but not limited to a small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound may be natural or synthetic.
As used herein, the term xe2x80x9ccomplementaryxe2x80x9d refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.
As used herein, the term xe2x80x9ccontactingxe2x80x9d means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention. The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc. Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoded by the nucleic acid, or fragment thereof.
As used herein, the phrase xe2x80x9chomologous nucleotide sequence,xe2x80x9d or xe2x80x9chomologous amino acid sequence,xe2x80x9d or variations thereof, refers to sequences characterised by a homology, at the nucleotide level or amino acid level, of at least the specified percentage. Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding other known wildtype go genes. Homologous amino acid sequences include those amino acid sequences which encode conservative amino acid substitutions, as well as polypeptides having neuropeptide binding and/or signalling activity. A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known wildtype GOs. Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein by reference in its entirety).
As used herein, the term xe2x80x9cisolatedxe2x80x9d nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
As used herein, the terms xe2x80x9cmodulatesxe2x80x9d or xe2x80x9cmodifiesxe2x80x9d means an increase or decrease in the amount, quality, or effect of a particular activity or protein.
As used herein, the term xe2x80x9coligonucleotidexe2x80x9d refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes.
As used herein, the term xe2x80x9cprobexe2x80x9d refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
By xe2x80x9camplificationxe2x80x9d it is meant increased numbers of DNA or RNA in a cell compared with normal cells. xe2x80x9cAmplificationxe2x80x9d as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, compared to the basal level.
As used herein, the phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d or xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes, primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at least about 60xc2x0 C. for longer probes, primers or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
The amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. The nucleotide sequences are presented by a single strand only, in the 5xe2x80x2 to 3xe2x80x2 direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letters code, or by the accepted single letter code.
xe2x80x9cVariantxe2x80x9d GO (or vGO) enzymes are those which have a substitution of at least one amino acid, and have superior enzymatic activity and/or thermostability when compared with the wildtype enzyme. The enzymatic activity may be improved with regard to the substrate methyl galactose, but alternatively may be improved with regard to other substrates. The particular substitutions are described by indicating the one letter code for the wildtype amino acid, followed by the amino acid position, followed by the substituted amino acid in the variant. The position number is determined counting the first amino acid of the mature sequence, the mature sequence being defined by the crystal structure as number 1, with each consecutive amino acid numbered consecutively. The pre and prosequences are not included in the mature sequence. For example, the V494A variant describes a variant GO having the wildtype""s valine at position 494 substituted with alanine. In cases where the wildtype""s amino acid may be replaced with one of two or more amino acids, the variants are described by adding the additional single letter codes preceded by a slash (/); e.g., Y436N/H indicates a valine at position 494 substituted by either asparagine or histidine. Thus two distinct variants are described in this example, namely Y436N and Y436H.
A polynucleotide of the invention must have a mutation which renders at least one codon xe2x80x9cnondegeneratexe2x80x9d, i.e., the codon must encode a different amino acid than the unmutated codon.
Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and is also intended to include allelic variants thereof. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or xe2x80x9cspliced out.xe2x80x9d RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a GO polypeptide, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts. Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation). While allelic variants of wildtype GO are not encompassed by the present invention, those allelic variants which have been mutated and which encode variant GOs of the invention are within the scope of the invention.
The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding vGO (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).
The wildtype GO nucleotide sequence is set forth in the following SEQ ID NO: I (gaoA, Genbank Accession No. M86819):
The corresponding amino acid sequence for the mature wildtype GO is set forth in the following SEQ ID NO: 2.
The sequence for the precursor protein, which includes the signal sequence and prosequence, may be found in Genbank Accession No. Q01745.
Several preferred DNA sequences of a mgo encoding a vGO polypeptide of the invention having superior enzymatic activity compared to wildtype are those which encode a variant GO having a substitution at any of positions C383, Y436, V494, and Q63. More preferably, the preferred polynucleotide encodes a variant GO having more than one of these preferred substitutions. More preferably, the polynucleotide encodes a variant GO having at least one of the substitutions selected from the group consisting of C383S, V494A, Q63K, and either Y436N or Y436H, more preferably at least two of those substitutions, more preferably at least three of those substitutions. A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA. These preferred examples are illustrative; the invention embraces other variants which have additional substitutions, and the polynucleotides that encode such variants, provided such variants have superior activity.
Several preferred DNA sequences of a mgo encoding a vGO polypeptide of the invention having superior enzymatic activity compared to wildtype are those which encode a variant GO having a substitution at any of positions G195, S553, G6, Q238, K342, N427, and Q63. More preferably, the preferred polynucleotide encodes a variant GO having more than one of these preferred substitutions. More preferably, the polynucleotide encodes a variant GO having at least one of the substitutions selected from the group consisting of S553C, G6R, Q238L, K342E, N427T, Q63K, and either G195A or G195E, more preferably at least two of those substitutions, more preferably at least three of those substitutions. In a preferred embodiment of the invention, the polynucleotide encodes a variant GO having at least one substitution conferring superior enzymatic activity and at least one substitution conferring superior thermostability. A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA. These preferred examples are illustrative; the invention embraces other variants which have additional substitutions, and the polynucleotides that encode such variants, provided such variants have superior thermostability. Preferred polypeptides include all those encoded by the preferred DNA sequences as described above.
The invention further embraces species homologs of the mgo DNA. For example, species homologs have been found in Gibberella fujikuroi, Polyporus circinatus, Arabidopsis thaliana (CAB65567), and Streptomyces coelicolor A3(2) (CAB41193), among others. Species homologs, sometimes referred to as xe2x80x9corthologs,xe2x80x9d in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with DNA of the invention. Generally, percent sequence xe2x80x9chomologyxe2x80x9d with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the preferred polynucleotides discussed above, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related vGO polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR). Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to vGO and structurally related polypeptides sharing one or more biological, enzymatic, immunological, and/or physical properties of vGO. Non-human species genes encoding proteins homologous to vGO can also be identified by Southern and/or PCR analysis. Knowledge of the sequence of a human mgo DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding vGO expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express vGO.
The disclosure herein of a full-length polynucleotide encoding a vGO polypeptide makes readily available to the worker of ordinary skill in the art every possible fragment of the full length polynucleotide which contains a mutation encoding the variant amino acid. The invention therefore provides fragments of vGO-encoding polynucleotides comprising at least 14, and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding vGO, provided that the fragment contains the mutated nucleotide codon. Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also may include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides (e.g., the wildtype), and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
Fragment polynucleotides are particularly useful as probes for detection of full-length or fragment mgo polynucleotides. One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding vGO, or used to detect variations in a polynucleotide sequence encoding vGO.
The invention also embraces DNAs encoding vGO polypeptides that hybridize under moderately stringent or high stringency conditions to the non-coding strand, or complement, of the preferred polynucleotides set forth above.
Exemplary highly stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1xc3x97 SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley and Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.
Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided. Expression constructs wherein vGO-encoding polynucleotides are operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided. Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and are typically selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell.
Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a vGO-encoding polynucleotide sequence.
According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner which permits expression of the encoded vGO polypeptide. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector. Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.
Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with a vGO of the present invention. Host cells of the invention are also useful in methods for the large-scale production of vGO polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
Knowledge of mgo DNA sequences allows for modification of cells to permit, or increase, expression of endogenous vGO. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring mgo promoter with all or part of a heterologous promoter so that the cells express vGO at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous vGO encoding sequences. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the mgo coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the mgo coding sequences in the cells.
The invention also provides purified and isolated vGO polypeptides encoded by a polynucleotide of the invention. Presently preferred are vGO polypeptides comprising at least one substitution at any of the positions S553, G6, Q238, K342, N427, Q63, and G195. More preferably, the vGO enzyme has a combination of substitutions, and more preferably the substitutions are selected from the group consisting of S553C, G6R, Q238L, K342E, N427T, Q63K, and either G195A or G195E.
The invention also embraces polypeptides that have at least 99%,at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention, provided that the sequence has at least the amino acid variations present in any of the preferred polypeptides set forth above. Percent amino acid sequence xe2x80x9cidentityxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the vGO sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence xe2x80x9chomologyxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the vGO sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.
In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment (Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference).
Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of vGO polypeptides are embraced by the invention.
The invention also embraces variant (or analog) vGO polypeptides. In one example, insertion variants are provided wherein one or more amino acid residues supplement a vGO amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the vGO amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.
Insertion variants include vGO polypeptides wherein one or more amino acid residues are added to a vGO amino acid sequence, or to an enzymatically active fragment thereof.
Other products of the invention also include mature vGO products, i.e., vGO products wherein leader or signal sequences are removed, with additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins. vGO products with an additional methionine residue at position xe2x88x921 (Metxe2x88x921-vGO) are contemplated, as are variants with additional methionine and lysine residues at positions xe2x88x922 and xe2x88x921 (Metxe2x88x922-Lysxe2x88x921-vGO). Variants of vGO with additional Met, Met-Lys, Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.
The invention also embraces vGO variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position xe2x88x921 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated.
Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of vGO is/are fused to another polypeptide.
In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a vGO polypeptide are removed. Deletions can be effected at one or both termini of the vGO polypeptide, or with removal of one or more non-terminal amino acid residues of vGO. Deletion variants, therefore, include all fragments of a vGO polypeptide.
The invention thus embraces polypeptide fragments of the preferred vGO polypeptides set forth above, wherein the fragments maintain enzymatic properties of a vGO polypeptide, provided the fragments contain the substituted amino acid(s) distinguishing the vGO polypeptide from the wildtype. Such fragments comprising at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of are comprehended by the invention. Preferred polypeptide fragments display antigenic properties unique to, or specific for, human vGO and its allelic and species homologs. Fragments of the invention having the desired enzymatic properties can be prepared by any of the methods well known and routinely practiced in the art.
In still another embodiment, the invention provides substitution variants of vGO polypeptides. Substitution variants include those polypeptides wherein one or more amino acid residues of a vGO polypeptide are removed and replaced with alternative residues, provided the substitution variants contain the variant amino acid(s) distinguishing the vGO polypeptide from the wildtype. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 1, 2, or 3 below.
Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1 (from WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.
Alternatively, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77) as set out in Table 2, immediately below.
As still another alternative, exemplary conservative substitutions are set out in Table 3, below.
It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells. Similarly, the invention further embraces vGO polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. Variants that display enzymatic properties of vGO and are expressed at higher levels are also contemplated.
In a related embodiment, the present invention provides compositions comprising purified polypeptides of the invention. Preferred compositions comprise, in addition to the polypeptide of the invention, an acceptable liquid, semisolid, or solid diluent that serves as a vehicle, excipient, or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.
With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode vGOs from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., xe2x80x9cMolecular cloning: a laboratory manualxe2x80x9d, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference in its entirety.
A nucleic acid molecule comprising any of the vGO nucleotide sequences described above may be obtained using any means of mutagenesis performed on the wildtype go sequence. Such methods include error prone PCR, use of mutagenic strains such as the XL1-Red mutator strain of E. coli (Stratagene Inc.), use of random mutagenesis methods involving mutagenic chemicals such as ethyl-methyl sulfonate (EMS) or involving irradiation by UV light or other radiations of higher or lower energy, combinatorial cassette mutagenesis (Delagrave, et al., (1993), Bio/Technology, 10, 1548-52), site-directed mutagenesis, mutagenesis by PCR involving the incorporation of one or more primers encoding mutations, mutagenesis by DNA shuffling (e.g., Stemmer, 1994, Nature, 370:389, and other closely related methods), and mutagenesis by any PCR method.
A nucleic acid molecule comprising any of the vGO nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein. See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis. PCR provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotides probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger), which is incorporated herein by reference in its entirety.
Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above. Vectors are used herein either to amplify DNA or RNA encoding vGO and/or to express DNA which encodes vGO. Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses. Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Other expression vectors include, but are not limited to, pSPORT vectors, pGEM vectors (Promega), pPROEXvectors (LTI, Bethesda, Md.), Bluescript vectors (Stratagene), pQE vectors (Qiagen), pSE420 (Invitrogen), and pYES2 (Invitrogen).
Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding vGO is operably linked or connected to suitable control sequences capable of effecting the expression of the vGO in a suitable host. DNA regions are operably linked or connected when they are functionally related to each other. For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence. Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, and sequences which control the termination of transcription and translation.
Preferred vectors preferably contain a promoter that is recognised by the host organism. The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacz promoters of E. coli and the SV40 early promoter (Benoist, et al. Nature, 1981, 290, 304-310, which is incorporated herein by reference in its entirety). Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.
Additional regulatory sequences can also be included in preferred vectors. Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgamo of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarno sequence may be directly followed by DNA encoding vGO and result in the expression of the mature vGO protein.
Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.
An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and vGO DNA. An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat. No. 4,399,216).
Nucleotide sequences encoding vGO may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al., Mol. Cell. Biol., 1983, 3, 280, Cosman et al., Mol. Immunol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
Another embodiment of the present invention is directed to transformed host cells having an expression vector comprising any of the nucleic acid molecules described above. Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell. Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.
If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. Preferably, eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973), which is incorporated herein by reference in its entirety).
In addition, a yeast host may be employed as a host cell. Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluyveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Shuttle vectors for replication in both yeast and E. coli are also included herein.
Alternatively, insect cells may be used as host cells. In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O""Rielly et al. (Eds.), W. H. Freeman and Company, New York, 1992, and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference in its entirety). In addition, the MAXBAC(trademark) complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for vGO or fragments thereof. Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fabxe2x80x2, F(abxe2x80x2)2, and FV, are also provided by the invention. The term xe2x80x9cspecific for,xe2x80x9d when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind vGO polypeptides exclusively (i.e., are able to distinguish vGO polypeptides from other known vGO polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between vGO and such polypeptides). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the vGO polypeptides of the invention are also contemplated, provided that the antibodies are specific for vGO polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
Non-human antibodies may be humanized by any of the methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. Antibodies of the invention are useful for, e.g., detecting or quantifying vGO, as well as purifying vGO. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is imnuunospecific.
In other embodiments of the invention, the polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins. Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypetides are used to capture interacting molecules. For example, molecules are incubated with the labeled polypeptides, washed to removed unbound polypeptides, and the polypeptide complex is quantified. Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.
Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors of GO, such as 6-thio-methyl-galactose identified as an inhibitor by Wachter et al., 1996, Biochemistry 35:14425 -14435, and others. In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.
The vGO enzyme may be used to oxidize compounds, such as, for example, hydroxyl containing compounds, provided that the group to be oxidized is available (e.g., sterically) for the enzyme to carry out its oxidation function. A group available to the enzyme in this regard is referred to herein as free of steric hindrance. Steric factors involved in the GO enzyme""s action are discussed generally in Cathmann et al., 1981, Biochem. Biophys. Res. Commun. 103:68-76, the disclosure of which is hereby incorporated by reference in its entirety. Examples of compounds for which vGO may be used to perform oxidation, and wherein the vGO may have improved enzymatic activity, include, but are not limited to, galactose, lactose, raffinose, dihydroxyacetone, diethylene glycol, ethanol and other primary alcohols, and guaran gums, D-galacto-hexodialdose, dihydroxyacetone, 3-hydroxy-2-oxo-propionaldehyde, glycerol, S(xe2x88x92)-glyceraldehyde, 6xe2x80x3-carboxyraffinose, methyl-alpha-D-galactopyranose, methyl-beta-D-galactopyranose, major glycolipid of human red cells, D-talose, 3-halo-1,2-propane-diols, GM1 ganglioside, D-galactosamine, melibiose, stachyose, desialyated glycoproteins (e.g., fetuin, mucin), N-acetyl-D-galactosamine, isopropyl-beta-D-thiogalactosylpyranoside, beta-thiodigalactoside, melibiitol, melibionic acid, 1,5-anhydrogalactitol, planteose, 2-glycerol-alpha-D-galactopyranoside, galactobiose, beta-D-galactopyranosyl, beta-D-galactopyranosyl, D-glucose, methyl-beta-D-thiogalactosylpyranoside, and the like. The GO enzyme has also been characterized as possessing activity as a superoxide dismutase (Cleveland et al., 1974, Biochim. Biophys. Acta 341:517-523).
For example, the oxidation of guar may be performed on a 1% guar mixture in pH 7 phosphate buffer containing galactose oxidase, catalase, and horse radish peroxidase. The reaction may be run at 26xc2x0 C. with constant mixing and sparging with air for approximately 3 hours. Suitable reaction conditions are set forth, for example, in U.S. Pat. No. 6,022,717, the disclosure of which is hereby incorporated herein by reference in its entirety.
The oxidized compounds resulting from interaction with the variant GO enzymes of the invention are useful in a wide variety of applications and chemical processes that will be readily apparent to those of skill in the art. Oxidized guar, for example, may be used in papermaking processes, such as those described generally in Smook, Handbook for Pulp and Paper Technologists (Canadian Pulp and Paper Assn. 1982), which is hereby incorporated by reference in its entirety.
Using the variants of the invention, one of skill in the art may more efficiently oxidize guar gum for use in, for example, a method of making paper. The variant enzyme is isolated in sufficient quantity, then used to oxidize guar gum, and the oxidized guar is added to the paper pulp during the paper-making process. More detailed descriptions of such papermaking processes may be found in U.S. Pat. No. 6,022,717.
There are many other possible uses of the variants of the invention, including, but not limited to, generation of H2O2 in situ; enzymatic synthesis of other aldehydes; pulp biobleaching; the use of galactose oxidase-Schiff""s reagent for early detection and prognosis in human colorectal adenocarcinoma (e.g., Carter et al., Clin Cancer Res 1997 September; 3(9):1479-89); and the use of galactose oxidase-glucan binding domain fusion proteins as targeting inhibitors of dental plaque bacteria (Lis and Kurimitsu. Antimicrob Agents Chemother 1997 May; 41(5):999-1003). Those of skill in the art will readily appreciate the many uses to which a galactose oxidase enzyme may be put.
Additional features of the invention will be apparent from the following Examples. Example 1 is actual, while the remaining Examples are prophetic.